Conformational and topological protein regulation

Abstract

Methods and compositions are provided for identifying novel conformers of proteins not known to exist as different conformers. By using chimeric genes replacing a native signal sequence with a different signal sequence resulting in the production of a conformer, one can compare the native protein with the product of the chimeric gene. Where the conformations are different, the different protein may be used for production of antibodies, elucidation of mechanisms associated with the native and different conformer protein, assays for the presence of the different conformer in physiological samples, identification of compounds specifically binding to the conformer, particularly drugs, etc. Where the formation of the conformer is associated with a diseased state, the conformer may be used in screens to identify compounds as drug candidates.

What is claimed is:  
     
       1. A method for detecting a conformational difference between proteins, said method comprising: 
       contacting a non-denatured protein, obtained by expression of a chimeric gene having a DNA sequence encoding at least substantially the same amino acid sequence as an open reading frame encoding a native protein, wherein said native protein is encoded by a gene comprising said open reading frame and a signal sequence native to said open reading frame, and wherein said chimeric gene has a signal sequence that is not native to said open reading frame so that a conformer of said native protein is produced, with an agent that detectably modifies accessible side groups of amino acids common to said conformer and to said native protein to produce a conformer and a native protein comprising modified side groups; and  
       detecting differences in said modified side groups between said conformer and said native protein as indicative of a conformational difference between said conformer and said native protein.  
     
     
       2. The method according to  claim 1 , wherein said agent is a crosslinking agent or a chemical modification agent. 
     
     
       3. The method according to  claim 1 , wherein said chemical modification agent is N-sulfo biotin or trinitrobenzenesulfonic acid. 
     
     
       4. The method according to  claim 1 , further comprising: 
       evaluating reactivity of said conformer to a protease, wherein altered reactivity is indicative of a conformational difference between said conformer and said native protein.